ARTICLE IN PRESS

1094-5539/$ - se

doi:10.1016/j.pu

Abbreviations

sulfoxide; DPPE

salt solution; 5-

methylpiperazin

{(2R,5R)-5-[1H

PKA, protein k�Correspond

Tel.: +358 3 35

E-mail addr

Pulmonary Pharmacology & Therapeutics 21 (2008) 222–233

www.elsevier.com/locate/ypupt

Histamine reverses IL-5-afforded human eosinophil survival byinducing apoptosis: Pharmacological evidence for a novel mechanism of

action of histamine

Hannele Hasalaa,c, Mark A. Giembyczb, Mirkka Janka-Junttilaa,c,Eeva Moilanena,c, Hannu Kankaanrantaa,d,�

aThe Immunopharmacology Research Group, Medical School/B, University of Tampere, FIN-33014 Tampere, FinlandbDepartment of Pharmacology and Therapeutics, Faculty of Medicine, Institute of Infection, Immunity and Inflammation,

University of Calgary, Calgary, Alberta, CanadacResearch Unit, Tampere University Hospital, Tampere, Finland

dDepartment of Respiratory Medicine, Tampere University Hospital, Tampere, Finland

Received 24 October 2006; received in revised form 8 March 2007; accepted 12 March 2007

Abstract

Eosinophils are essential inflammatory cells in the pathogenesis of asthma and atopic conditions. Histamine, released from mast cells

and basophils in response to allergen exposure, is a critical mediator in the allergic response. Histamine exerts its effects via four

unequivocally characterized histamine receptors, H1�4. Several functions of eosinophils have previously been shown to be stimulated by

histamine. However, its effects on eosinophil apoptosis are unknown. The aim of the present study was to resolve the effects of histamine

on constitutive apoptosis of human eosinophils and on the survival-enhancing action of interleukin (IL)-5. Additional experiments were

conducted to elucidate the histamine receptor(s) involved in any response seen and the associated signal transduction cascade.

Human isolated peripheral blood eosinophils were cultured in the absence or presence of histamine, IL-5 and receptor antagonists/

agonists or mediator inhibitors/analogues. Apoptosis was assessed by measuring the relative DNA content of propidium iodide (PI)-

stained cells and the effects were confirmed by morphological analysis with bright field microscopy. Caspase activities were assessed by

using commercial Caspase-Glos 3/7, 8 and 9 luminescence assays.

Histamine ð102100mMÞ partially reversed IL-5-induced human eosinophil survival by enhancing apoptosis as assessed by measuring the

relative DNA content of PI-stained cells. This effect was not mediated through any of the known histamine receptors or through non-

specific activation of 5-hydroxytryptamine receptors or a-adrenoceptors. Moreover, the reversal of IL-5-inhibited eosinophil apoptosis by

histamine seemed not to utilize the conventional intracellular second-messenger pathways including cyclic AMP, protein kinase A or

phospholipase C. Inhibition of caspase 6 and caspases 1, 10 or 12 reversed the effects of histamine but also inhibited apoptosis in general.

In conclusion, the data presented herein indicate that histamine induces human eosinophil apoptosis in the presence of a survival-

prolonging cytokine by a mechanism that does not apparently involve the activation of any of the currently known histamine receptor

subtypes. The possibility exists that another, as yet unidentified, histamine receptor may exist in human eosinophils that regulates

survival, although the participation of histamine receptor-independent mechanisms cannot be excluded.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Histamine; Histamine receptors; Eosinophils; Apoptosis; IL-5; Caspases

e front matter r 2007 Elsevier Ltd. All rights reserved.

pt.2007.03.002

: AC, adenylyl cyclase; cAMP, cyclic adenosine-30; 50-monophosphate; CI, confidence interval; DAG, diacylglycerol; DMSO, dimethyl

, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamide, HIC-antagonist; HIC, intracellular histamine ‘‘receptor’’; HBSS, Hank’s balanced

HT, 5-hydroxytryptamine; IL, interleukin; IP3, inositol(1,4,5)trisphosphate; JNJ7777120, 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-

e, H4-antagonist; MMPCI, 7-methyl-2-[(4-methylpiperazin-1-yl)carbonyl]-1H-indole, H4-antagonist; OUP-16, (-)-2-cyano-1-methyl-3-

-imidazol-4(5)-yl]tetrahydrofuran-2-yl}methylguanidine, H4-agonist; PI, propidium iodide; PIP2, phosphatidylinositol(4,5)bisphosphate;

inase A; PKC, protein kinase C; PLC, phospholipase C; PTX, pertussis toxin

ing author. The Immunopharmacology Research Group, Medical School/B, University of Tampere, FIN-33014 Tampere, Finland.

51 7318; fax: +358 3 3551 8082.

ess: blhaka@uta.fi (H. Kankaanranta).

ARTICLE IN PRESSH. Hasala et al. / Pulmonary Pharmacology & Therapeutics 21 (2008) 222–233 223

1. Introduction

Eosinophilic inflammation represents an essential ele-ment in the pathogenesis of allergic diseases such as asthmaand allergic rhinitis [1–3]. The balance between eosinophilmaturation, recruitment and removal largely determinesthe number of eosinophils in the blood and tissues [3,4].Apoptosis, or programmed cell death, is a controlledprocess of cell suicide that allows the removal of senescentcells or those whose continued survival would be detri-mental for the organism. Furthermore, failure in theapoptotic process of eosinophils has been associated withpulmonary and allergic diseases [3,5]. Apoptosis is char-acterized by specific morphological and biochemicalchanges such as cell shrinkage, nuclear coalescence,chromatin condensation and endonuclease-catalyzedDNA breakdown followed by fragmentation of the cellinto apoptotic bodies, which are phagocytozed intactwithout further induction of inflammation [6,7]. Apoptosisand its induction is regarded as an important mechanism inthe resolution of eosinophilic inflammation [3,5,6] and ithas been shown that eosinophil apoptosis is delayed inpatients with asthma, inhalant allergy and atopic derma-titis [8,9]. In vitro, eosinophil apoptosis is inhibited bycytokines such as interleukin (IL)-3, IL-5 and granulocyte/macrophage colony-stimulating factor [1,3].

Histamine is an important physiological amine thatplays a major role in many physiological and pathologicalprocesses. Histamine was originally considered a mediatorof acute inflammatory and immediate hypersensitivityreactions but is now believed to also affect chronicinflammatory processes and to regulate the immuneresponse [10,11]. The effects of histamine are mediatedthrough at least four distinct cell surface, G-protein-coupled receptors (histamine H1, H2, H3 and the newlyidentified H4Þ [12–17] and eosinophils have been suggestedto express all of these subtypes [10,11]. A fifth histaminereceptor, located intracellularly and denoted by histamineHIC, has also been described in hematopoietic cells [18].Histamine binds to the different receptor subtypes withmarkedly different affinity: the K i-values range from5–15 nM for the histamine H3 and H4 receptor subtypes[10,11,19] to approximately 102100mM for histamine H1,H2 and HIC [10,11,18,19]. The four cell surface histaminereceptor subtypes transduce extracellular signals by using avariety of G-proteins and intracellular second-messengersystems. Through the Gq=11-coupled histamine H1 receptor,histamine is thought to produce the symptoms and signs ofallergy via stimulation of phospholipase C (PLC),which catalyzes the breakdown of phosphatidylinosi-tol(4,5)bisphosphate ðPIP2Þ to diacylglycerol (DAG) andinositol(1,4,5)trisphosphate ðIP3Þ that, in turn, activateprotein kinase C (PKC) and mobilize Ca2þ from intracel-lular stores, respectively [10,11]. The histamine H2 receptoris positively coupled through Gs to adenylyl cyclase (AC)and mediates the formation of cyclic adenosine-30; 50-monophosphate (cAMP). The histamine H2 receptor is

traditionally associated with mediating the enhancement ofgastric acid secretion but has recently also been suggestedto participate in the suppression of inflammation [10,11].The histamine H3 and H4 receptors are both coupled toGi=o and inhibit AC activity, suppress the formation ofcAMP and the activity of protein kinase A (PKA).The histamine H3 receptor is mainly expressed in thenervous system and controls the release of histamineand other neurotransmitters, whereas the histamine H4

receptor has been suggested to be involved in immuneregulatory functions such as chemotaxis and cytokinesecretion [10,11].Histamine affects several essential functions of eosino-

phils. Allergen-induced accumulation of eosinophils in theairways, nose and skin can be inhibited by histamine H1

receptor antagonists [10,11]. Histamine also affects eosi-nophil migration: histamine, at high concentrations, waspreviously suggested to inhibit eosinophil chemotaxis viathe histamine H2 receptor, whereas at low concentrations,histamine was suggested to enhance chemotaxis via thehistamine H1 receptor [10,11]. However, it has recentlybeen suggested that the newly identified histamine H4

receptor is responsible for the selective recruitment andchemotaxis of eosinophils [20]. In addition, through thehistamine H4 receptor, histamine has also been found tomediate cell shape change, upregulation of adhesionmolecules and to induce actin polymerization andintracellular calcium mobilization in eosinophils [21,22].Furthermore, histamine inhibits eosinophil degranu-lation via stimulation of the histamine H2 receptorsand a consequent elevation of intracellular levels ofcAMP [23].The effects of histamine on granulocyte apoptosis

remain scantly studied. Histamine, at high concentrations,has been suggested to induce human neutrophil apoptosiswhich could be reversed by histamine H1 and H2 receptorantagonists as well as with a pan-caspase inhibitor and aninhibitor of PKC [24]. There are no published data on theeffect of histamine on human eosinophil apoptosisalthough it has been suggested that histamine does notaffect eosinophil survival in the absence of cytokines [25]. Ithas, however, been described that the antihistamines (aka

histamine H1 receptor antagonists) oxatomide [26], fex-ofenadine [27] and diphenhydramine and chlorpheniramine[28] promote human eosinophil apoptosis whereas ketoti-fen [29] has been shown to induce primary necrosis inhuman eosinophils. The results describing that histamineH1 receptor antagonists influence human eosinophilsurvival suggest that histamine itself may also have a rolein the regulation of human eosinophil apoptosis. Thus,studies on the effects of histamine on human eosinophilsare warranted.The objective of the present study described herein was

to resolve whether histamine affects constitutive apoptosisor IL-5-induced survival of human eosinophils and, if so,to elucidate the receptor(s) and signalling pathwayinvolved.

ARTICLE IN PRESSH. Hasala et al. / Pulmonary Pharmacology & Therapeutics 21 (2008) 222–233224

2. Materials and methods

2.1. Eosinophil isolation and culture

One hundred milliliters of peripheral venous blood wasobtained from volunteers with normal or slightly elevatedblood eosinophil counts. The volunteers were healthy oratopic. Subjects with hypereosinophilic syndrome wereexcluded. Before donating blood, the subjects gave writteninformed consent to the study protocol approved by theEthics Committee of Tampere University Hospital (Tam-pere, Finland). Eosinophils were isolated under sterileconditions to 499% purity by using CD16-negativeimmunomagnetic bead selection as previously described[8,30–32]. Isolated eosinophils were cultured for 40 h(þ37 �C, 5% CO2) in the absence or presence of histamine,IL-5 and other selected compounds as indicated in RPMI1640 medium (Dutch modification) with 10% fetal bovineserum and antibiotics unless otherwise stated.

2.2. Determination of apoptosis by the relative DNA content

assay by flow cytometry

The number of apoptotic cells was assessed by measuringthe relative DNA content by flow cytometry (FACScan,Becton Dickinson, San Hose, CA) of propidium iodide(PI)-stained cells [8, 30–32]. Endonuclease-catalyzed DNAfragmentation is regarded as a specific feature of apoptosis[33]. Therefore, the cells showing decreased relative DNAcontent were considered to be apoptotic. Eosinophils weresuspended in 300ml of hypotonic PI solution (25mgml�1 in0.1% sodium citrate and 0.1% Triton X-100), protectedfrom light and incubated at þ4 �C for 1 h before flowcytometric analysis.

2.3. Morphological analysis

To confirm the mode of cell death, eosinophil morphol-ogy was assessed by bright field microscopy. For morpho-logical analysis, eosinophils were spun onto cytospin slides(500 revmin�1, 5min) and stained with May-Grunwald–Giemsa after fixation in methanol. Cells showing thecharacteristic apoptotic morphology, such as cell shrink-age, nuclear coalescence and chromatin condensation, wereconsidered to have undergone apoptosis.

2.4. Caspase activity assay

Caspase 3/7, 8 and 9 activities in eosinophils wereassessed by using Caspase-Glos 3/7, 8 and 9 assays(Promega Corp., Madison, USA) according to themanufacturer’s instructions. Briefly, human eosinophilswere cultured in RPMI 1640 medium (Dutch modification)with 10% fetal bovine serum and antibiotics in the absenceand presence of IL-5 (1 pM) and histamine ð100mMÞ for16 h. Equal volumes of Caspase-Glos 3/7, 8 or 9 reagents

were added after which the samples were incubated for 1 hat room temperature before measurement of luminescence.

2.5. Materials

Histamine, mepyramine (pyrilamine maleate), cimeti-dine, thioperamide maleate, Rp-8-CPT-cAMPS, 8-Br-cAMP, methysergide maleate, 5-hydroxytryptamine(5-HT) hydrochloride, phentolamine hydrochloride,(�)-adrenaline hydrochloride, PI, JNJ7777120 (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine), dexa-methasone and pertussis toxin (PTX) were purchased fromSigma Chemical Co. (St. Louis, MO, USA).Z-D(OMe)QMD(OMe)-FMK, Z-VEID-FMK, Ac-IETD-CHO, Ac-LEHD-CHO and Q-VD-OPh were obtainedfrom Merck (Darmstadt, Germany). Other reagents wereobtained from the following sources: Z-Asp-CH2-DCB(Peptide Institute, Inc., Osaka, Japan), Caspase-Glos 3/7,8, 9 assays (Promega Corp., Madison, WI, USA), cirazo-line hydrochloride, clonidine hydrochloride, D609,U73122, N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethana-mide fumarate (DPPE fumarate) and clobenpropit dihy-drobromide (Tocris Cookson Ltd., Avonmouth, UK), anti-CD16 microbeads and the magnetic cell separation system(Miltenyi Biotec, Bergish Gladbach, Germany), humanrecombinant IL-5 (R&D system Europe, Abingdon, UK),Ficoll-Paque (Pharmacia AB, Uppsala, Sweden), antibio-tics and RPMI 1640 (Dutch modification) (Gibco BRL,Paisley, UK), fetal bovine serum (Euroclone, Pero, Italy),Hank’s balanced salt solution (HBSS) and RPMI 1640(BioWhittaker, Verviers, Belgium), May-Grunwald(Merck, Darmstadt, Germany) and Giemsa (J.T. Baker,Deventer, Holland). (-)-2-Cyano-1-methyl-3-{(2R,5R)-5-[1H-imidazol-4(5)-yl]tetrahydrofuran-2-yl}methylgua-nidine (OUP-16) was a kind gift from Prof. AtsushiYamatodani and Shinya Harusawa from the Universityof Osaka, Japan. H4-antagonist 7-methyl-2-[(4-methylpi-perazin-1-yl)carbonyl]-1H-indole (MMPCI) was a kind giftfrom Dr. Michael Peck from UCB Pharma (Brussels,Belgium).Histamine, mepyramine, cimetidine, thioperamide,

5-HT, D609, adrenaline, cirazoline and clonidine weredissolved in RPMI 1640 medium (Dutch modification).Clobenpropit, DPPE, Rp-8-CPT-cAMPS, 8-Br-cAMP anddexamethasone were dissolved in HBSS. OUP-16,MMPCI, JNJ7777120, U73122, methysergide and allcaspase inhibitors were dissolved in dimethyl sulfoxide(DMSO). The final concentration on DMSO in culture was0.5% except for Z-Asp-CH2-DCB for which the finalconcentration of DMSO within the cells was 1.0%. The0.5–1% DMSO was not found to affect eosinophil viabilityas assessed by bright field microscopy (n ¼ 6, data notshown). Similar concentrations of DMSO were added tothe control cultures. Stock solution of phentolamine wasprepared in ethanol. The final concentration of ethanol inthe culture was 0.1%, and a similar concentration ofethanol was added to the control cultures. The 0.1%

ARTICLE IN PRESSH. Hasala et al. / Pulmonary Pharmacology & Therapeutics 21 (2008) 222–233 225

ethanol did not affect eosinophil viability as assessed byflow cytometry (n ¼ 4, data not shown). PTX wasdissolved in 50% (v/v) glycerol containing 50mM Tris,pH 7.5, 10mM glycine and 0.5M NaCl. Similar solutionwas also added to the control, and was not found to affecteosinophil viability as assessed by flow cytometry (n ¼ 5,data not shown).

2.6. Statistics

The results are expressed as the mean� SEM. Apoptosisis expressed as apoptotic index (number of apoptotic cells/total number of cells, i.e. apoptotic index 0.1 means that10% of the cells are apoptotic). Statistical significance wascalculated by analysis of variance for repeated measuressupported by the Dunnett test or by paired t-tests by usingGraphPad Instat software (GraphPad Software, SanDiego, CA, USA). Differences were considered statisticallysignificant when po0:05. The concentration–response dataof histamine-induced apoptosis, the EC50 (effective con-centration that produces 50% of the maximal effect) valueand the 95% confidence interval (CI) were analyzed byusing GraphPad Prism software (GraphPad Software, SanDiego, CA, USA).

3. Results

3.1. Effect of histamine on human eosinophil apoptosis

3.1.1. Constitutive apoptosis

Human eosinophils cultured for 40 h in cytokine-deprived conditions underwent spontaneous apoptosis(apoptotic index 0:66� 0:07) as defined by relative DNAfragmentation. When different concentrations ð0:012

100mMÞ of histamine were added to culture, no alterationsin the rate of apoptosis were seen (n ¼ 6, Fig. 1a).Histamine ð100mMÞ consistently had no effect on eosino-phil apoptosis in the absence of cytokines when apoptosiswas assessed by bright field microscopy (apoptotic indiceswere 0:50� 0:07 and 0:41� 0:06 with and without hista-mine, respectively, n ¼ 6, p40:05).

3.1.2. IL-5-induced eosinophil survival

Culture of human eosinophils for 40 h in the presence of1 pM IL-5 markedly enhanced eosinophil survival andattenuated the apoptotic index to 0:15� 0:02. Histamineð0:012100mMÞ partially reversed IL-5-induced eosinophilsurvival by enhancing apoptosis (n ¼ 6, Fig. 1b, e and f).The enhancement of human eosinophil apoptosis byhistamine was concentration dependent (EC50 ¼ 0:56mM½CI ¼ 0:3320:97mM�, Fig. 1c insert) and reached amagnitude of approximately 40% at concentrations ofhistamine between 10 and 100mM (Fig. 1c). The magnitudeof the apoptosis-promoting effect of histamine in thepresence of IL-5 was similar to the effect of dexamethasone(n ¼ 4, Fig. 1d). The mode of cell death was confirmed bybright field microscopy and 100mM histamine was found to

increase eosinophil apoptosis by 56% in the presence of1 pM IL-5. The apoptotic indices were 0:08� 0:02 and0:13� 0:07 in the absence and presence of histamineð100 mMÞ, respectively (n ¼ 6, po0:01).Similar to the apoptosis-enhancing effect of glucocorti-

coids, which is abolished by higher concentrations ofcytokines [3], the effect of histamine in reversing IL-5-mediated eosinophil survival also fell off when theconcentration of IL-5 was increased 10-fold. In thepresence of 10 pM IL-5, 10mM histamine increasedeosinophil apoptosis by approximately 40% but this effectwas not quite statistically significant due to highervariability (n ¼ 6, p40:05, data not shown). Thus,subsequent experiments were performed by using the lower(1 pM) concentration of IL-5.

3.2. Role of caspases in histamine-induced human eosinophil

apoptosis

3.2.1. Caspase activities

To assess the role of caspases in histamine-inducedreversal of IL-5-afforded eosinophil survival, the activitiesof caspases 3/7, 8 and 9 were measured by using Caspase-Glos 3/7, 8 and 9 assays. Active caspases 3/7, 8 and 9 weredetected after 16 h incubation. Activities of all caspases 3/7,8 and 9 were markedly reduced by 1 pM IL-5 (Fig. 2).Histamine ð100mMÞ was found to slightly increase theactivities of caspases 3/7 and 9 in the presence of 1 pM IL-5(Fig. 2).

3.2.2. Effect of caspase inhibitors on histamine induced

apoptosis

To further evaluate the role of different caspases inhistamine ð100mMÞ-induced eosinophil apoptosis, theeffects of various caspase inhibitors in the absence andpresence of IL-5 (1 pM) were evaluated. Unexpectedly, apan-caspase inhibitor, Z-Asp-CH2-DCB ð200mMÞ, furtherenhanced histamine-induced apoptosis in the presence ofIL-5 (Fig. 3), whereas a broad-range inhibitor of caspases1, 3, 8, 9, 10 and 12, Q-VD-OPh ð20mMÞ, reversedhistamine-induced apoptosis in the presence of IL-5. Themechanism of this phenomenon remains obscure. Ananalogous effect to Q-VD-OPh was found with the caspase6 inhibitor Z-VEID-FMK ð200mMÞ (Fig. 3), i.e. reversal ofhistamine-induced apoptosis. In contrast, inhibitors ofcaspases 3 (Z-DQMD-FMK, 200mM), 8 (Ac-IETD-CHO,100mM) and 9 (Ac-LEHD-CHO, 100mM) did not exertany effects on eosinophil apoptosis (Fig. 3). In addition,Q-VD-OPh and Z-VEID-FMK reduced apoptosis inhistamine-treated cells in the absence of IL-5 (Fig. 3).

3.3. Role of histamine receptors in histamine-induced human

eosinophil apoptosis

3.3.1. Histamine H1;H2 and H3 receptor subtypes

Pretreatment of eosinophils for 15min with mepyramine(1mM, a histamine H1 receptor antagonist—K i�1 nM

ARTICLE IN PRESS

Fig. 1. Effect of histamine on human eosinophil apoptosis. The apoptotic indices of eosinophils cultured with histamine in the absence (a) and presence (b)

of 1 pM IL-5 are shown. Percentage changes in apoptosis by different histamine concentrations in the presence (c) of 1 pM IL-5. In (c), the insert shows the

concentration–response curve of histamine-induced apoptosis. In (d), the effect of dexamethasone on constitutive and IL-5-inhibited eosinophil apoptosis

is shown for comparison. The flow cytometry histograms of one experiment representative of six essentially identical experiments of 1 pM IL-5-induced

eosinophil survival (e) and 100mM histamine-induced partial reversal of 1 pMIL-5-mediated eosinophil survival (f) are also shown. The figure in the top

right corner (e,f) represents the percentage of apoptotic cells. Apoptosis was analyzed by the relative DNA fragmentation assay. Each data point (a–d)

represents the mean � SEM of n ¼ 426 independent determinations using eosinophils from different donors. ** indicates po0:01 as compared with the

respective control.

H. Hasala et al. / Pulmonary Pharmacology & Therapeutics 21 (2008) 222–233226

[34]), cimetidine (10mM, a histamine H2 receptor antago-nist—K i�0:8mM [34]) and thioperamide (10 nM, a hista-mine H3 receptor antagonist—K i�10 nM [19]) or different

combinations of these antagonists had no effect on thesurvival-enhancing activity of IL-5 (1 pM) in the absence orpresence of histamine ð100mMÞ (Fig. 4). Similarly, when

ARTICLE IN PRESSH. Hasala et al. / Pulmonary Pharmacology & Therapeutics 21 (2008) 222–233 227

the antagonists were used alone or in combinations, nosignificant effects on constitutive apoptosis or apoptosis inthe presence of only histamine were observed (p40:05,data not shown) except for the combination of thioper-

Fig. 2. Effect of histamine ð100mMÞ on caspase 3/7, 8 and 9 activation in

human eosinophils in the presence of IL-5 ð1 pMÞ after a 16 h incubation.

Caspase activities were measured with Caspase-Glos 3/7, 8 and 9 assays.

Each data point represents the mean � SEM of n ¼ 6 independent

determinations using eosinophils from different donors. * indicates

po0:05, ** indicates po0:01 and ns indicates p40:05.

Fig. 3. Effect of caspase inhibitors on histamine-induced eosinophil apoptosis.

absence of IL-5 (1 pM) and different caspase inhibitors. Apoptosis was analyze

mean � SEM of n ¼ 6 independent determinations using eosinophils from di

p40:05 as compared with the respective control (1%DMSO for Z-Asp-CH2-D

pan-caspase inhibitor; Q-VD-OPh, a broad-spectrum inhibitor of caspases 1, 3

caspase 6 inhibitor; Ac-IETD-CHO, a caspase 8 inhibitor; Ac-LEHD-CHO, a

amide and cimetidine, which slightly inhibited spontaneouseosinophil apoptosis (po0:05, data not shown).

3.3.2. The histamine H4 receptor subtype

Thioperamide is also an antagonist of the histamine H4

receptor at concentrations in the low micromolar rangeðK i�0:13mMÞ [13,16,17,19] and was examined on eosino-phil apoptosis in the absence and presence of histamineð100 mMÞ and IL-5 (1 pM). As shown in Fig. 5a,thioperamide did not reverse the effect of histamine butslightly attenuated the rate of apoptosis in general at thehighest concentration ð10 mMÞ studied (Fig. 5a).To further investigate the role of the H4 subtype, the

effect of JNJ7777120, a recently described selectivehistamine H4 receptor antagonist ðK i�4 nMÞ [35], wasstudied on human eosinophil apoptosis in the absence andpresence of histamine and IL-5. At concentrations ofJNJ7777120 up to 500 nM, apoptosis of human eosinophilswas unaffected (Fig. 5b). To exclude the possibility that thehigh concentration of histamine ð100mMÞ used in theseexperiments would, by competition, limit the degree ofantagonism produced by JNJ7777120, we also tested theeffect of 10mM JNJ7777120 on eosinophil apoptosisinduced by 10mM histamine in the presence of 1 pM IL-5. Again, under this experimental condition, the effect ofhistamine was not reversed (p40:05, data not shown).Similar results were obtained with another selectivehistamine H4 receptor antagonist, MMPCI ðpA2 ¼ 7:5Þ,which is selective for the histamine H4 receptor up toconcentrations of 10mM (Michael Peck, UCB Pharma,personal communication). Thus, MMPCI did not re-verse the apoptosis-promoting effect of histamine onIL-5-afforded eosinophil survival (Fig. 5c). To eliminate

Percentage changes in apoptosis by histamine ð100mMÞ in the presence and

d by the relative DNA fragmentation assay. Each data point represents the

fferent donors. * indicates po0:05, ** indicates po0:01 and ns indicates

CB and 0.5%DMSO for the other caspase inhibitors). Z-Asp-CH2-DCB, a

, 8, 9, 10 and 12; Z-DQMD-FMK, a caspase 3 inhibitor; Z-VEID-FMK, a

caspase 9 inhibitor.

ARTICLE IN PRESS

Fig. 4. Effect of histamine H123 receptor antagonists on histamine-induced eosinophil apoptosis. The apoptotic indices of eosinophils cultured with IL-5

(1 pM), with or without histamine ð100mMÞ and a combination of histamine H123 receptor antagonists. Apoptosis was analyzed by the relative DNA

fragmentation assay. Each data point represents the mean � SEM of n ¼ 6 independent determinations using eosinophils from different donors. **

indicates po0:01 as compared with the respective control.

H. Hasala et al. / Pulmonary Pharmacology & Therapeutics 21 (2008) 222–233228

the possibility that histamine at 100mM competed outthe antagonist, the effect of MMPCI ð10mMÞ on10mM histamine-induced eosinophil apoptosis in thepresence of 1 pM IL-5 was studied and found not toreverse the pro-apoptotic effect of histamine (p40:05, datanot shown).

To confirm that the histamine H4 receptor was notresponsible for the apoptosis-promoting effect of histamineon IL-5-induced survival of human eosinophils, two othersets of experiments were conducted. Thus, OUP-16, ahistamine H4 receptor agonist [36], and clobenpropit,an H3 receptor antagonist with partial agonist action atthe H4 receptor at concentrations above 1 nM[12,13,16,21], were examined to establish if they couldpromote apoptosis in a manner similar to histamine.However, neither OUP-16 ð0:01210mMÞ nor clobenpropit(0.1–30 nM) affected apoptosis in the absence or presenceof IL-5 (n ¼ 527, p40:05, data not shown) at concentra-tions that spanned their estimated K i’s ðOUP-16 ¼ 125 nM[37]; clobenpropit ¼ 8 nM [19]).

3.3.3. Effect of a histamine HIC ‘‘receptor’’ antagonist on

human eosinophil apoptosis

Histamine has also been described to have an intracel-lular binding site that has been denoted the histamine HIC

receptor, which is activated by micromolar concentrationsof histamine [18]. To determine if histamine exerts itsapoptosis-promoting effects via the intracellular histaminereceptor, we tested the effect of DPPE, a putativeantagonist of the histamine HIC receptors [18], oneosinophil apoptosis in the absence and presence ofhistamine ð100mMÞ and IL-5 (1 pM). DPPE did not reverse

the histamine-induced apoptosis (n ¼ 6, p40:05, data notshown) although spontaneous eosinophil apoptosis wasslightly decreased as well as apoptosis seen in the presenceof 100mM histamine without IL-5 (n ¼ 6, po0:01, data notshown).

3.4. Role of different signal transduction pathways in

histamine-induced human eosinophil apoptosis

Although none of the currently defined histaminereceptor subtypes apparently mediated the ability ofhistamine to promote apoptosis of eosinophils cultured inthe presence of IL-5, additional studies were performed togain information that would support a receptor-mediatedmechanism.

3.4.1. Effect of PTX on histamine-induced apoptosis

Pretreatment of eosinophils with PTX, which inactivatesGi=o, at concentrations 0.1, 0.5 and 1:0 mgml�1 for 90 minat þ37 �C, did not influence human eosinophil apoptosisunder any conditions examined (Fig. 6a).

3.4.2. Role of cAMP and PKA—pathway in histamine-

induced apoptosis

The cell permeant cAMP analogue, 8-Br-cAMP ð1mMÞ,significantly and markedly decreased constitutive eosino-phil apoptosis (n ¼ 4, po0:001, data not shown) but hadno effect on cell survival in the presence of IL-5 (n ¼ 4,p40:05, data not shown). Therefore, the effect ofhistamine on IL-5-mediated eosinophil survival was notmimicked by a cAMP analogue, indicating it was not dueto an increase in the levels of cAMP in the cell. In addition,

ARTICLE IN PRESSH. Hasala et al. / Pulmonary Pharmacology & Therapeutics 21 (2008) 222–233 229

the PKA inhibitor, Rp-8-CPT-cAMPS, had no effect oneosinophil apoptosis in the absence or presence ofhistamine (100mM) and IL-5 (1 pM; Fig. 6b).

Fig. 5. Effect of histamine H4 receptor antagonists on histamine-induced

eosinophil apoptosis. (a) The apoptotic indices of eosinophils cultured

with different concentrations of thioperamide in the absence and presence

of histamine and IL-5. (b) The apoptotic indices of eosinophils cultured

with different concentrations of JNJ7777120 in the absence and presence

of histamine and IL-5. (c) The apoptotic indices of eosinophils cultured

with different concentrations of MMPCI in the absence and presence of

histamine and IL-5. Apoptosis was analyzed by the relative DNA

fragmentation assay. Each data point represents the mean � SEM of

n ¼ 6 independent determinations using eosinophils from different

donors. * indicates po0:05 and ** indicates po0:01 as compared with

the respective control.

Fig. 6. Role of different signal transduction pathways in human eosinophil

apoptosis. (a) The apoptotic indices of eosinophils cultured with pertussis

toxin (PTX) in the absence and presence of histamine and IL-5. PTX

disrupts Gi=o-protein function. (b) The apoptotic indices of eosinophils

cultured with PKA inhibitor Rp-8-CPT-cAMPS in the absence and presence

of histamine and IL-5. (c) The apoptotic indices of eosinophils cultured with

PLC inhibitors D609 or U73122 in the absence and presence of histamine

and IL-5. Apoptosis was analyzed by the relative DNA fragmentation assay.

Each data point represents the mean � SEM of n ¼ 425 independent

determinations using eosinophils from different donors. *** indicates

po0:001 as compared with the respective control.

ARTICLE IN PRESSH. Hasala et al. / Pulmonary Pharmacology & Therapeutics 21 (2008) 222–233230

3.4.3. Role of PLC in histamine-induced apoptosis

To evaluate the possible role of PLC in humaneosinophil apoptosis, we tested two different pharmacolo-gical inhibitors, D609 and U73122. D609 had no effects onhuman eosinophil apoptosis whereas U73122 inhibitedconstitutive eosinophil apoptosis and decreased apoptosisin the presence of histamine without IL-5. However, in IL-5-treated cells, U73122 did not reverse the apoptosis-enhancing effect of histamine (Fig. 6c).

3.5. Role of 5-HT-receptors and a-adrenoceptors in

histamine-induced human eosinophil apoptosis

As relatively high concentrations of histamine wererequired to promote eosinophil apoptosis, we investigatedwhether other amine receptors, namely those activated by5-HT and catecholamines, could be involved in the actionof histamine. Thus, we first examined the effect ofmethysergide, a nonselective 5-HT1-, 5-HT2- and 5-HT7-receptor antagonist, as well as the natural ligand for thesereceptors, 5-HT, on eosinophil apoptosis. Methysergide(1210 mM) did not alter the rate of histamine-inducedeosinophil apoptosis either in the absence or presence ofIL-5 (n ¼ 5, p40:05, data not shown). Furthermore, 5-HT(12100 mM) did not mimic the apoptosis-promoting effectof histamine on IL-5-afforded eosinophil survival (n ¼ 4,p40:05, data not shown).

Phentolamine ð10mMÞ, a nonselective a-adrenoceptorantagonist, slightly decreased constitutive eosinophil apop-tosis in the absence and presence of histamine (n ¼ 4,po0:05, data not shown). However, no reversal of theenhancement of apoptosis by histamine in the presence ofIL-5 was observed (n ¼ 4, p40:05, data not shown). Tofurther exclude the involvement of a-adrenoceptors inmediating histamine-induced human eosinophil apoptosis,we tested the effects of adrenaline (0:121mM, a non-selective agonist), cirazoline (1210 mM, a selective a1-agonist) and clonidine (102100mM, an a2-agonist) onapoptosis of human eosinophils in the absence andpresence of IL-5. None of these pharmacological interven-tions increased apoptosis (n ¼ 4, p40:05, data not shown)although a slight decrease in eosinophil survival in theabsence of IL-5 was observed at high (100mM) concentra-tions of clonidine (n ¼ 4, po0:05, data not shown).

4. Discussion

In the present study we have shown that histamine partlyreverses IL-5-afforded survival of human eosinophils byinducing apoptosis. This effect of histamine is notapparently mediated through any of the known histaminereceptors or through agonism at 5-HT-receptors or a-adrenoceptors. Moreover, the reversal of IL-5-inhibitedeosinophil apoptosis by histamine does not utilize thecommon intracellular second-messenger pathways includ-ing cAMP, PKA or PLC but may involve caspase 6 and atleast some of caspases 1, 10 or 12. Thus, the results raise

the possibility that human eosinophils may express a novel,hitherto unidentified, histamine receptor.The increase in eosinophil apoptosis elicited by hista-

mine in the presence of IL-5 reached a magnitude of 40%.This degree of reversibility is comparable to that producedby glucocorticoids, which is considered one of their keyanti-inflammatory mechanisms in the treatment of eosino-philic conditions such as asthma [3,38]. However, hista-mine did not increase constitutive eosinophil apoptosis(unlike glucocorticoids), which implies that the mechanismof action differs between these two classes of compounds.The concentration of IL-5 ð1 pM � 30 pgml�1Þ used in thepresent study and reversed by histamine is quite lowalthough very potent in increasing eosinophil survival.Recent reports indicate that the plasma concentration ofIL-5 is approximately 4 pgml�1 in healthy individuals [39],whereas the sputum concentrations of IL-5 are approxi-mately 30 and 90 pgml�1 in healthy and asthmatic persons,respectively [40]. Thus, the used concentration of IL-5 isclinically relevant.Previous studies have reported that histamine concen-

trations ranging from 10nM to 10mM are required toachieve the histamine H4 receptor-mediated effects oneosinophil chemotaxis, cell shape change, upregulation ofadhesion molecules and intracellular calcium mobilization[20–22]. Furthermore, even higher concentrations ofhistamine have been reported to be necessary for theenhancement of apoptosis of neutrophils [24]. In addition,the histamine concentrations used in nasal [41,42] andinhalation [43] challenges are manifold as compared to theeffective concentration used in this study. Therefore, itseems likely that levels of histamine similar to those used toinduce apoptosis of cytokine-treated human eosinophils in

vitro can be achieved locally after histamine challenges usedin the diagnosis of asthma and allergic rhinitis, which maybe relevant also in vivo. Moreover, during inflammation,the local concentration of histamine released from baso-phils and mast cells has been estimated to be in themillimolar range acting on neighboring cells such aseosinophils in an autocrine and paracrine fashion [44].Taken together, this potentially anti-inflammatory actionof histamine on cytokine-afforded eosinophil survivaloccurs at clinically relevant concentrations and themagnitude of this effect is equivalent to that evoked byglucocorticoids. Thus, we suggest that the reversal of IL-5-induced eosinophil survival by histamine represents a novelmeans of regulating the immune system. The response tohistamine seen in vivo will be derived from the net effect ofthose receptor subtypes that are activated, of whichsome effects are pro-inflammatory and others may beanti-inflammatory. It is possible that all histamine receptorsubtypes are activated on a variety of cells during theallergic reaction and that blockage of the possible novelhistamine receptor on eosinophils may enhance inflamma-tion further.Our data suggest that the ability of histamine to partially

reverse IL-5-afforded inhibition of human eosinophil

ARTICLE IN PRESSH. Hasala et al. / Pulmonary Pharmacology & Therapeutics 21 (2008) 222–233 231

apoptosis is not mediated through any of the currentlyknown histamine receptors. Given the diverse role ofhistamine through the newly identified histamine H4

receptor in the regulation of inflammation [37], wehypothesized that the effect of histamine could behistamine H4 receptor-mediated. However, experimentswith selective agonists and antagonists failed to provideevidence that this effect of histamine was mediated throughthe histamine H4 receptor subtype under the experimentalconditions used. Similar studies with selective receptorantagonists also excluded a role of the histamine H1, H2,H3 and HIC receptors. The histamine H1 and H2 receptorantagonists, mepyramine and cimetidine, were used atconcentrations well in excess of their respective affinities atthe H1 and H2 receptors [34] but lower than theconcentrations likely to promote eosinophil apoptosis dueto mechanisms unrelated to histamine receptor antagonism[26–28]. If either the H1 or the H2 receptor subtype wasinvolved in the reversal of IL-5-mediated eosinophilsurvival, mepyramine or cimetidine would have beenexpected to antagonize the effect of histamine, which wasused at a concentration approximately similar to its K i atthe H1 and H2 receptors [19]. Were the histamine H4

receptor subtype involved, significant agonism would havebeen predicted with clobenpropit and OUP-16 based ontheir affinities at the histamine H4 receptor [19,37].Moreover, at least some antagonism by thioperamide,JNJ7777120 and MMPCI would have been expected at theH4 receptor [19,35] even though histamine was used with amuch higher concentration than its affinity for the H4

receptor subtype [19]. In previous studies evaluating the H4

receptor-mediated effects of histamine on eosinophils, theeffects of similar histamine concentrations were antago-nized with similar thioperamide and JNJ7777120 concen-trations and agonized with similar clobenpropitconcentrations as used in the present study [21,22].Thus, the present results indicate that the histamine H4

receptor subtype does not mediate the apoptosis-enhancingeffect of histamine on IL-5-induced human eosinophilsurvival.

Regarding the histamine H3 receptor subtype, significantantagonism of histamine-induced apoptosis may not haveoccurred with thioperamide due to competition [19].However, evidence suggesting the existence of the hista-mine H3 receptors on human eosinophils is now knownto be incorrect [1,45,46]. Indeed, the novel histaminereceptor discovered in eosinophils by Raible and co-workers in the 1990s [45,46] has now been identifiedas the H4 subtype [12,13,16,17,20,21,37,47]. Moreover,human eosinophils have recently been shown not to expressthe histamine H3 receptor [48]. Thus, histamine-inducedhuman eosinophil apoptosis in the presence of IL-5 isunlikely to be mediated through the histamine H3 receptorsubtype.

As relatively high concentrations of histamine wererequired to promote apoptosis of IL-5-treated eosinophils,the possibility that histamine may act through other amine

receptors was evaluated. In particular, given the possiblerole of 5-HT in asthma [49,50] and because 5-HT has beensuggested to act as an eosinophil chemoattractant via the 5-HT2A-receptor [51], we explored whether 5-HT-receptorscould mediate the pro-apoptotic effect of histamine oneosinophils. However, the 5-HT-receptor antagonist,methysergide, did not block histamine-induced eosinophilapoptosis nor did 5-HT mimic the effects of histamine. Therole of a-adrenoceptors was also evaluated. Neitherselective (cirazoline, clonidine) nor nonselective (adrena-line) a-adrenoceptor agonists emulated the effect ofhistamine. Phentolamine, an a-adrenergic antagonist, didnot reverse the apoptosis-enhancing effect of histamine onIL-5-mediated human eosinophil survival either. Addi-tional studies, performed to determine if common intra-cellular second-messenger signalling pathways couldaccount for the ability of histamine to promote apoptosis,excluded a role for PTX-sensitive pathways, cAMP, PKAand PLC.It has been proposed that additional histamine receptors

remain to be discovered as not all effects of histamine canyet be explained by the currently defined subtypes [47]. Ourresult demonstrating the partial reversal of cytokine-afforded survival of human eosinophils by histaminethrough induction of apoptosis represents yet anothereffect of histamine that cannot be explained by any of thecurrently known histamine receptors or by activation ofother amine receptors on eosinophils. These data furthersuggest the possible existence of an additional receptor forhistamine although a histamine receptor-independentmechanism cannot be excluded. Multiple other contingen-cies also exist that may mediate histamine-inducedenhancement of human eosinophil apoptosis in thepresence of IL-5. For instance, histamine may, in someunknown way, modify the signalling cascades activated byIL-5 in human eosinophils. Although the detailed signal-ling pathways involved in cytokine-induced eosinophilsurvival are not fully understood, cytokine receptors formdimers and phosphorylate tyrosine residues upon activa-tion. The subsequent signalling events include adaptorproteins, Ras, mitogen-activated protein kinase pathways,and Janus kinase, signal transducer and activator oftranscription pathways [3]. Histamine might also stimulatethe production of another mediator or cytokine thatinterferes with IL-5-mediated eosinophil survival. Forexample, prostaglandin D2 [52] and IL-4 [53] could besuch mediators as they have been reported to induceeosinophil apoptosis. Histamine may also promote eosi-nophil apoptosis through a non-histamine receptor that isnot a 5-HT or alpha variant nor is a classical 7-spanG-protein-coupled receptor. For example, an ion-channelreceptor or a nuclear receptor might be mediating thehistamine effect. Alternatively, histamine may utilize non-receptor-mediated mechanisms. Thus, detailed additionalexperiments focusing on the mechanism of histamine-induced reversal of IL-5-afforded inhibition of humaneosinophil apoptosis are highly warranted.

ARTICLE IN PRESSH. Hasala et al. / Pulmonary Pharmacology & Therapeutics 21 (2008) 222–233232

Regulation of caspase activity is considered essential inthe apoptotic process. Caspases are constitutively expressedcysteine proteases that cleave substrates at specific asparticacid residues. In the resting cell, caspases are normallypresent as inactive proenzymes that are activated aftercleavage at internal aspartate residues to form the matureprotein [54]. During apoptosis, caspases are activated in anamplifying cascade and can be divided into two functionallydifferent subgroups, the initiator and effector caspases [54].At present, the specific caspase pathways mediatingeosinophil apoptosis remain poorly characterized [3].However, the presence of caspases 3, 6, 7, 8 and 9 hasbeen described in eosinophils and spontaneous eosinophilapoptosis seems to utilize these caspases [3]. Recently,caspases 3 and 6 have been suggested to play a role inorazipone-induced human eosinophil apoptosis [55]. Thepresent results suggest that caspase 6 and at least some ofcaspases 1, 10 or 12 may mediate the apoptosis-promotingeffect of histamine on IL-5-treated human eosinophils. Incontrast, caspases 3 and 9 seem to be unimportant inhistamine-induced reversal of cytokine-afforded eosinophilsurvival despite their activation during apoptosis.

In interpreting the current results, the existence ofdifferent subpopulations of eosinophils is a possibility alsoworth mentioning. For instance, a small proportion ofeosinophils have been shown to express CD16 uponactivation in allergic patients [56]. Thus, during eosinophilisolation with the CD16-negative selection method, we mayhave lost this part of the eosinophil population. Whetherthese cells have different responses to histamine or possessa different histamine receptor expression profile than thosein the present study remains to be elucidated.

In conclusion, our results demonstrate that histaminepartially reverses IL-5-afforded survival of human eosino-phils by inducing apoptosis and the magnitude of this effectmirrors that produced by glucocorticoids. The pro-apoptotic action of histamine occurs at clinically relevantconcentrations and represents a novel mechanism ofregulation within the immune system. The effect ofhistamine on eosinophil apoptosis cannot be explained byany of the currently known histamine receptors or by non-specific activation of other amine receptors on eosinophils.Thus, although we cannot exclude the possibility thathistamine-induced apoptosis occurs through a receptor-independent mechanism, the data presented herein im-plicate the participation of a novel histamine receptorsubtype.

Acknowledgments

The financial support obtained from Tampere Tubercu-losis Foundation (Tampere, Finland), the Finnish Anti-Tuberculosis Association Foundation (Helsinki, Finland),the Academy of Finland (Helsinki, Finland), the MedicalResearch Fund of Tampere University Hospital (Tampere,Finland) and the Canadian Institutes for Health Research

(Canada) is gratefully acknowledged. The authors appreci-ate the secretarial assistance of Mrs. Heli Maatta.

References

[1] Giembycz MA, Lindsay MA. Pharmacology of the eosinophil.

Pharmacol Rev 1999;51:213–340.

[2] Howarth PH. Eosinophils and rhinitis. Clin Exp Allergy Rev 2005;

5:55–63.

[3] Kankaanranta H, Moilanen E, Zhang X. Pharmacological regulation

of human eosinophil apoptosis. Curr Drug Targets Inflamm Allergy

2005;4:433–45.

[4] Simon HU, Yousefi S, Schranz C, Schapowal A, Bachert C, Blaser K.

Direct demonstration of delayed eosinophil apoptosis as a mechan-

ism causing tissue eosinophilia. J Immunol 1997;158:3902–8.

[5] Bianchi SM, Dockrell DH, Renshaw SA, Sabroe I, Whyte MK.

Granulocyte apoptosis in the pathogenesis and resolution of lung

disease. Clin Sci 2006;110:293–304.

[6] Haslett C. Granulocyte apoptosis and its role in the resolution and

control of lung inflammation. Am J Respir Crit Care Med

1999;160:S5–11.

[7] Ohta K, Yamashita N. Apoptosis of eosinophils and lymphocytes in

allergic inflammation. J Allergy Clin Immunol 1999;104:14–21.

[8] Kankaanranta H, Lindsay MA, Giembycz MA, Zhang X, Moilanen

E, Barnes PJ. Delayed eosinophil apoptosis in asthma. J Allergy Clin

Immunol 2000;106:77–83.

[9] Wedi B, Raap U, Lewrick H, Kapp A. Delayed eosinophil

programmed cell death in vitro: a common feature of inhalant

allergy and extrinsic and intrinsic atopic dermatitis. J Allergy Clin

Immunol 1997;100:536–43.

[10] Akdis CA, Simons FER. Histamine receptors are hot in immuno-

pharmacology. Eur J Pharmacol 2006;533:69–76.

[11] Jutel M, Blaser K, Akdis CA. Histamine in allergic inflammation and

immune modulation. Int Arch Allergy Immunol 2005;137:82–92.

[12] Liu C, Ma X, Jiang X, Wilson SJ, Hofstra CL, Blevitt J, et al.

Cloning and pharmacological characterization of a fourth histamine

receptor (H(4)) expressed in bone marrow. Mol Pharmacol 2001;

59:420–6.

[13] Morse KL, Behan J, Laz TM, West REJ, Greenfeder SA, Anthes JC,

et al. Cloning and characterization of a novel human histamine

receptor. J Pharmacol Exp Ther 2001;296:1058–66.

[14] Nakamura T, Itadani H, Hidaka Y, Ohta M, Tanaka K. Molecular

cloning and characterization of a new human histamine receptor,

HH4R. Biochem Biophys Res Commun 2000;279:615–20.

[15] Nguyen T, Shapiro DA, George SR, Setola V, Lee DK, Cheng R,

et al. Discovery of a novel member of the histamine receptor family.

Mol Pharmacol 2001;59:427–33.

[16] Oda T, Morikawa N, Saito Y, Masuho Y, Matsumoto S. Molecular

cloning and characterization of a novel type of histamine receptor

preferentially expressed in leukocytes. J Biol Chem 2000;275:36781–6.

[17] Zhu Y, Michalovich D, Wu H, Tan KB, Dytko GM, Mannan IJ,

et al. Cloning, expression, and pharmacological characterization of a

novel human histamine receptor. Mol Pharmacol 2001;59:434–41.

[18] MacGlashan Jr. D. Histamine: a mediator of inflammation. J Allergy

Clin Immunol 2003;112:S53–9.

[19] Lim HD, van Rijn RM, Ling P, Bakker RA, Thurmond RL, Leurs R.

Evaluation of histamine H1-, H2-, and H3-receptor ligands at the

human histamine H4 receptor: identification of 4-methylhistamine as

the first potent and selective H4 receptor agonist. J Pharmacol Exp

Ther 2005;314:1310–21.

[20] O’Reilly M, Alpert R, Jenkinson S, Gladue RP, Foo S, Trim S, et al.

Identification of a histamine H4 receptor on human eosinophils—role

in eosinophil chemotaxis. J Recept Signal Transduct Res 2002;22:

431–48.

[21] Buckland KF, Williams TJ, Conroy DM. Histamine induces

cytoskeletal changes in human eosinophils via the H(4) receptor. Br

J Pharmacol 2003;140:1117–27.

ARTICLE IN PRESSH. Hasala et al. / Pulmonary Pharmacology & Therapeutics 21 (2008) 222–233 233

[22] Ling P, Ngo K, Nguyen S, Thurmond RL, Edwards JP, Karlsson L,

et al. Histamine H4 receptor mediates eosinophil chemotaxis with cell

shape change and adhesion molecule upregulation. Br J Pharmacol

2004;142:161–71.

[23] Ezeamuzie CI, Philips E. Histamine H(2) receptors mediate the

inhibitory effect of histamine on human eosinophil degranulation. Br

J Pharmacol 2000;131:482–8.

[24] Hur J, Kang MK, Park JY, Lee SY, Bae YS, Lee SH, et al. Pro-

apoptotic effect of high concentrations of histamine on human

neutrophils. Int Immunopharmacol 2003;3:1491–502.

[25] Levi-Schaffer F, Temkin V, Malamud V, Feld S, Zilberman Y. Mast

cells enhance eosinophil survival in vitro: role of TNF-alpha and

granulocyte-macrophage colony-stimulating factor. J Immunol

1998;160:5554–62.

[26] Domae M, Sagara H, Sakaue M, Fukuda T, Kamikawa Y. The

antiallergic drug oxatomide promotes human eosinophil apoptosis

and suppresses IL-5-induced eosinophil survival. J Allergy Clin

Immunol 2003;111:567–72.

[27] Vancheri C, Mastruzzo C, Tomaselli V, Bellistri G, Pistorio MP,

Greco LR, et al. The effect of fexofenadine on expression of

intercellular adhesion molecule 1 and induction of apoptosis on

peripheral eosinophils. Allergy Asthma Proc 2005;26:292–8.

[28] Hasala H, Moilanen E, Janka-Junttila M, Giembycz MA, Kankaan-

ranta H. First-generation antihistamines diphenhydramine and

chlorpheniramine reverse cytokine-afforded eosinophil survival by

enhancing apoptosis. Allergy Asthma Proc 2007;28:79–86.

[29] Hasala H, Malm-Erjefalt M, Erjefalt J, Giembycz MA, Zhang X,

Moilanen E, et al. Ketotifen induces primary necrosis of human

eosinophils. J Ocular Pharmacol Ther 2005;21:318–27.

[30] Kankaanranta H, De Souza PM, Barnes PJ, Salmon M, Giembycz

MA, Lindsay MA. SB 203580, an inhibitor of p38 mitogen-activated

protein kinase, enhances constitutive apoptosis of cytokine-deprived

human eosinophils. J Pharmacol Exp Ther 1999;290:621–8.

[31] Kankaanranta H, De Souza PM, Giembycz MA, Lindsay MA.

Human eosinophil isolation and the measurement of apoptosis. In:

Chung KF, Adcock IM, editors. Methods in molecular medicine:

asthma, vol. 44. London: Humana press; 2000. p. 99–110.

[32] Zhang X, Moilanen E, Lahti A, Hamalainen M, Giembycz MA,

Barnes PJ, et al. Regulation of eosinophil apoptosis by nitric oxide:

role of c-Jun-N-terminal kinase and signal transducer and activator

of transcription 5. J Allergy Clin Immunol 2003;112:93–101.

[33] Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A

rapid and simple method for measuring thymocyte apoptosis by

propidium iodide staining and flow cytometry. J Immunol Methods

1991;139:271–9.

[34] Leurs R, Smit MJ, Timmerman H. Molecular pharmacological

aspects of histamine receptors. Pharmacol Ther 1995;66:413–63.

[35] Thurmond RL, Desai PJ, Dunford PJ, Fung-Leung WP, Hofstra CL,

Jiang W, et al. A potent and selective histamine H4 receptor

antagonist with anti-inflammatory properties. J Pharmacol Exp Ther

2004;309:404–13.

[36] Hashimoto T, Harusawa S, Araki L, Zuiderveld OP, Smit MJ,

Imazu T, et al . A selective human H(4)-receptor agonist: (-)-2-

cyano-1-methyl-3-[(2R,5R)-5- [1H-imidazol-4(5)-yl]tetrahydrofuran-

2-y] methylguanidine. J Med Chem 2003;46:3162–5.

[37] de Esch IJ, Thurmond RL, Jongejan A, Leurs R. The histamine H4

receptor as a new therapeutic target for inflammation. Trends

Pharmacol Sci 2005;26:462–9.

[38] Druilhe A, Letuve S, Pretolani M. Glucocorticoid-induced apoptosis

in human eosinophils: mechanisms of action. Apoptosis 2003;8:

481–95.

[39] Park YJ, Oh EJ, Park JW, Kim M, Han K. Plasma eosinophil

cationic protein, interleukin-5, and ECP/Eo count ratio in patients

with various eosinophilic diseases. Ann Clin Lab Sci 2006;36:262–6.

[40] Park SW, Jangm HK, An MH, Min JW, Jang AS, Lee JH, et al.

Interleukin-13 and interleukin-5 in induced sputum of eosinophilic

bronchitis: comparison with asthma. Chest 2005;128:1921–7.

[41] Plevkova J, Brozmanova M, Pecova R, Tatar M. The effects of nasal

histamine challenge on cough reflex in healthy volunteers. Pulm

Pharmacol Ther 2006;19:120–7.

[42] Wuestenberg EG, Hauswald B, Huettenbrink KB. Thresholds in

nasal histamine challenge in patients with allergic rhinitis, patients

with hyperreflectory rhinopathy, and healthy volunteers. and healthy

volunteers, Am J Rhinol 2004;18:371–5.

[43] Koskela HO, Kontra KM, Purokivi MK, Randell JT. Hypertonicity

of the challenge solution may increase the diagnostic accuracy of

histamine challenge. Respir Med 2005;99:726–34.

[44] Adams GK, Lichtenstein L. In vitro studies of antigen-induced

bronchospasm: effect of antihistamine and SRS-A antagonist on

response of sensitized guinea pig and human airways to antigen.

J Immunol 1979;122:555–62.

[45] Raible DG, Schulman ES, DiMuzio J, Cardillo R, Post TJ. Mast cell

mediators prostaglandin-D2 and histamine activate human eosino-

phils. J Immunol 1992;148:3536–42.

[46] Raible DG, Lenahan T, Fayvilevich Y, Kosinski R, Schulman ES.

Pharmacologic characterization of a novel histamine receptor on

human eosinophils. Am J Respir Crit Care Med 1994;149:1506–11.

[47] Hough LB. Genomics meets histamine receptors: new subtypes, new

receptors. Mol Pharmacol 2001;59:415–9.

[48] Nakayama T, Kato Y, Hieshima K, Nagakubo D, Kunori Y,

Fujisawa T, et al. Liver-expressed chemokine/CC chemokine ligand

16 attracts eosinophils by interacting with histamine H4 receptor.

J Immunol 2004;173:2078–83.

[49] Barnes PJ. Histamine and serotonin. Pulm Pharmacol Ther 2001;

14:329–39.

[50] Cazzola I, Matera MG. 5-HT modifiers as a potential treatment of

asthma. Trends Pharmacol Sci 2000;21:13–6.

[51] Boehme SA, Lio FM, Sikora L, Pandit TS, Lavrador K, Rao SP, et

al. Cutting edge: serotonin is a chemotactic factor for eosinophils and

functions additively with eotaxin. J Immunol 2004;173:3599–603.

[52] Ward C, Dransfield I, Murray J, Farrow SN, Haslett C, Rossi AG.

Prostaglandin D2 and its metabolites induce caspase-dependent

granulocyte apoptosis that is mediated via inhibition of IkBadegradation using peroxisome proliferator-activated receptor-g-in-dependent mechanism. J Immunol 2002;168:6232–43.

[53] Wedi B, Raap U, Lewrick H, Kapp A. IL-4-induced apoptosis

in peripheral blood eosinophils. J Allergy Clin Immunol 1998;102:

1013–20.

[54] Thornberry NA. Caspases: key mediators of apoptosis. Chem Biol

1998;5:R97–103.

[55] Kankaanranta H, Ilmarinen P, Zhang X, Nissinen E, Moilanen E.

Antieosinophilic activity of orazipone. Mol Pharmacol 2006;69:1861–70.

[56] Davoine F, Lavigne S, Chakir J, Ferland C, Boulay ME, Laviolette

M. Expression of FcgRIII (CD16) on human peripheral blood

eosinophils increases in allergic conditions. J Allergy Clin Immunol

2002;109:463–9.